JP2021089286A - Integrating nanopore sensors within microfluidic channel arrays using controlled breakdown - Google Patents

Abstract

To provide a device for fabricating nanopore sensors by controlled breakdown.SOLUTION: An apparatus for fabricating nanopores in a membrane composed of a dielectric membrane includes: a first substrate 14 with a microchannel 4 to be used in common formed on an exposure surface; a support 16 arranged on the exposure surface and configured to hold a membrane 12; a second substrate 15 with one or more microfluidic channels 5 formed on an inner surface, the second substrate being disposed on the support with the inner surface facing the support such that the one or more microfluidic channels are fluidly separated by the membrane from the common microchannel; and a pair of electrodes including a reference electrode arranged on one side of the membrane and two or more additional electrodes arranged on the opposite side of the membrane. The pair of electrodes generates an electric potential across the membrane. The two or more additional electrodes are disposed on the membrane so that the electric field may be uniform across the membrane.SELECTED DRAWING: Figure 1

Description

Cross-reference to related applications This application claims the priority benefit of US Provisional Application No. 62 / 094,669 filed December 19, 2014. The entire disclosure of the above application is incorporated herein by reference.

The present disclosure relates to the fabrication of nanopore sensors in microfluidic channels by controlled fracture (CBD) in solid membranes.

Nanopores are now a class of well-established unlabeled sensors capable of electrically detecting a single molecule. This technique relies on applying voltages to both sides of a nanoscale opening in a thin insulating film immersed in an ionic solution, and the resulting ionic current fluctuations are electrophoretically driven through the nanopores. It can be associated with the migration of individual charged biomolecules such as DNA and proteins. These changes in conductance provide information about the length, size, charge, and shape of the moving molecule. This technique is particularly attractive because of its wide range of single molecule studies, including DNA sequencing, protein detection and unfolding, single molecule mass spectrometry and force spectroscopy.

Nanopores may be formed by incorporating protein pores into a lipid bilayer membrane or may be made into a thin solid membrane. Biological pores can have extremely low noise properties, but the very brittle lipid bilayer membranes traditionally used as supports limit the life of the pores and the voltage that can be applied, thus limiting some. Uses are limited. Solid nanopores, on the other hand, are more durable under a wider range of experimental conditions such as applied voltage, temperature and pH, and their size can be adjusted in place. In general, solid nanopores are easier to integrate into a robust lab-on-a-chip device as an array. In fact, recent research has revealed various integration strategies for embedding such nanopores in microfluidic networks. The nanopores used in these studies are generally constructed on very thin (10 nm-50 nm) dielectric films (eg SiN) using high energy ion or electron beams. However, the fabrication of nanopores using FIB or TEM poses a problem in terms of integration. Since perforation with an energy particle beam requires straight-ahead access, it is required to fabricate nanopores prior to integration in microfluidic devices. This results in tighter alignment requirements for both nanopore fabrication and device assembly, resulting in smaller microfluidic channel dimensions and single membrane array formation, especially to minimize electrical noise. This raises the issue of limiting the yield of functional devices. More generally, these traditional nanofabrication techniques rely on nanopore fabrication in a vacuum environment, which inevitably leads to handling risks and wetness when moving to aqueous solutions for biosensing experiments. Will lead to problems.

Recently, another method for producing solid nanopores with high reliability using a high electric field has been proposed, and this is referred to as nanopore production by controlled fracture (CBD) in the present specification. Under supporting complete insulating film and common biological sensing experimental conditions (eg, in 1M KCl), the dielectric breakdown event is induced in the insulating film, resulting in a small diameter of 1 nm but a large size with an accuracy of less than 1 nm. A single adjustable nanopore is formed. The simplicity of the CBD method is sufficient for the integration of nanopore sensors in complex microfluidic architectures and for future lab-on-a-chip devices. By combining the advanced sample handling and processing capabilities inherent in microfluidic devices with in-situ nanopore fabrication, it is expected that various problems associated with integration will be alleviated and the range of applications of sensing platforms will be expanded. Further details regarding this fabrication technique are apparent from US Patent Application Publication No. 2015/010808, wherein the title of the invention is "Fabrication of Nanopores Using High Electric Fields" and the entire content is incorporated herein by reference.

This section provides background information regarding this disclosure, which is not necessarily prior art.

This section provides a high-level overview of the disclosure and is not a comprehensive disclosure of its full scope or all its features.

A device for making one or more nanopores on the membrane is presented. The device includes a first substrate with commonly used microchannels formed on the exposed surface, a support arranged on the exposed surface of the first substrate and configured to hold the membrane, and inside. A second substrate in which one or more microfluidic channels are formed on the surface of the inner surface so that the one or more microfluidic channels are fluidly separated from the microchannels commonly used by the membrane. It includes a second substrate arranged on the support with its surface facing the support and a set of electrodes that generate a potential difference on both sides of the membrane. A set of electrodes includes a reference electrode located on one side of the membrane and two or more additional electrodes located on the opposite side of the membrane, with the two or more additional electrodes being the entire membrane. It is arranged with respect to the membrane so that the electric field is uniform. In some embodiments, the potential difference on both sides of the membrane produces an electric field with a value greater than 0.1 volt / nanometer.

The device is electrically connected to one of the electrodes and can operate to measure the current flowing between one of one or more microfluidic channels and a commonly used microchannel. Further including a current sensor and a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of a pore and responds to the detection of the sudden increase in the measured current. Then, the potential difference generated on both sides of the membrane is eliminated.

In one embodiment, two or more additional electrodes were placed in a first electrode located upstream of the membrane in one or more microfluidic channels and in one or more microfluidic channels downstream of the membrane. Includes a second electrode.

In some embodiments, a plurality of microfluidic channels are formed on the inner surface of the second substrate. Each microchannel has a set of associated electrodes. In this way, an array of nanopores (corresponding to the number of microfluidic channels) can be made into the membrane.

In other embodiments, the film may be placed directly on the first substrate without the use of a support. In such an embodiment, the set of electrodes may include two reference electrodes arranged in a commonly used microchannel such that one is upstream of the membrane and the other is downstream of the membrane.

In another aspect of the present disclosure, the intermediate layer is placed directly on the support and thus between the support and the second substrate. In this case, the device for producing one or more nanopores on the film is arranged on the exposed surface of the first substrate and the exposed surface of the first substrate in which commonly used microchannels are formed on the exposed surface. A support configured to hold the membrane, an intermediate layer placed on the support with at least one via formed, and a second with one or more microfluidic channels formed on the inner surface. A first substrate that is placed on an intermediate layer with its inner surface facing the support so that one or more microfluidic channels are fluidly separated from the microfluidic channels commonly used by the membrane. Includes 2 substrates and a pair of electrodes arranged on the front and back of the film. The pair of electrodes creates a potential difference on both sides of the membrane. One or more vias in the intermediate layer are configured to fluidly connect one or more microfluidic channels to the exposed surface of the membrane to generate a uniform electric field in and around the vias. In some embodiments, the potential difference on both sides of the membrane produces an electric field with a value greater than 0.1 volt / nanometer.

The device is electrically connected to one of the electrodes and can operate to measure the current flowing between one of one or more microfluidic channels and a commonly used microchannel. Further including a current sensor and a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of a pore and responds to the detection of the sudden increase in the measured current. Then, the potential difference generated on both sides of the membrane is eliminated.

In some embodiments, a plurality of microfluidic channels are formed on the inner surface of the second substrate. Each microchannel has a set of associated electrodes. In this way, an array of nanopores (corresponding to the number of microfluidic channels) can be made into the membrane.

In other embodiments, the film may be placed directly on the first substrate without the use of a support. In such an embodiment, the set of electrodes may include two reference electrodes arranged in a commonly used microchannel such that one is upstream of the membrane and the other is downstream of the membrane.

In yet another aspect of the present disclosure, one or more microfluidic channels run adjacent to the membrane so as to generate a uniform electric field over the entire region of the membrane, thus reducing the number of electrodes required. To. In one embodiment, the microfluidic channel forms a loop downstream of the electrode placed in the channel, with a portion of the loop passing over the membrane.

In some embodiments, one or more control valves are arranged in the microfluidic channel and operate to control the amount of flow through the microfluidic channel. The control valve may be implemented by an elastomeric polymer that is fluidly coupled to an pneumatic source and operated by this pneumatic source.

In some embodiments, a plurality of microfluidic channels are formed on the inner surface of the second substrate. Each microchannel has a set of associated electrodes. In this way, an array of nanopores (corresponding to the number of microfluidic channels) can be made into the membrane. Also, each microfluidic channel in the array of microfluidic channels passes through a portion of the membrane, with at least two control valves located internally, one valve located upstream of the membrane, and the other valve of the membrane. It is located downstream. In this way, the value of the potential difference on both sides of the membrane is controlled by adjusting the flow through the control valves arranged in the array of microfluidic channels.

Further available areas will be apparent from the description provided herein. The descriptions and examples in this summary are for illustration purposes only and are not intended to limit the scope of this disclosure.

The drawings described herein are for illustration purposes only, and are not intended to limit the scope of the present disclosure, nor are they intended to limit the scope of the present disclosure.

Throughout the drawings, the corresponding reference marks indicate the corresponding parts.

FIG. 1 is a schematic view of an apparatus for producing nanopores, in which one electrode is inserted on one side of the membrane. 2A and 2B are cross-sectional views of an exemplary embodiment of a device having five independent microfluidic channels, each on a device having five microfluidic channels placed directly on the membrane. The reflection optical image taken from is shown. 3A to 3C are schematic views showing an example of an assembly method of the apparatus shown in FIG. 2A. 4A-4C are schematics showing different electrode arrangements that can be used to generate a uniform electric field over the surface region of the film. 5A and 5B are cross-sectional views of a second exemplary embodiment of an apparatus having a microvia layer, five that are placed directly on top of the membrane but separated from the membrane by the microvia layer. The catoptric image taken from above the device having the microfluidic channel of. 6A and 6B are images showing finite element modeling of the electric field in devices with and without microfluidic vias, respectively. 6C and 6D are magnified images of the electric field surrounding the nanopores shown in FIGS. 6A and 6B, respectively. FIG. 6E is a graph showing the magnitude of the electric field measured along a surface in the middle of a SiN film having a thickness of 20 nm when a voltage of 10 V is applied (as in the case of nanopore fabrication). FIG. 6F is a graph showing the magnitude of the electric field of the device without micro vias. 7A and 7B show (a) leakage current through the SiN film seconds before nanopore formation by CBD at 10V, and (b) conductance for five nanopores independently made on a single 5-channel device. It is a graph which shows the current-voltage (IV) curve used to estimate the nanopore diameter using the based model. 8A and 8B show (a) power spectral density (PSD) noise comparisons, (b) current traces for macro cells (black), 5-channel devices (blue), and 5-channel devices with microvias (red). It is a graph which shows. All measurements were performed with no voltage applied and no nanopores made, sampled at 250 KHz and low-pass filtered at 100 kHz with a 4-pole Bessel filter in 1 M KCl at pH 7.5. 9A and 9B show (a) human α-thrombin detection at an applied voltage of -200 mV using a pore of 10.5 nm, (b) -200 mV (black square), -250 mV (red triangle), -300 mV. Normalized average current block (0% indicates fully open pores, 100% indicates fully blocked pores) and total for 10 kb dsDNA transfer through 11.5 nm pores in (blue circles). It is a scatter diagram which shows the event time of. Each data point represents a single event. The inset shows a temporary current block as biomolecules interact with nanopores. For clarity, the inset figure multiplied the data by -1. 10A and 10B are cross-sectional views of an exemplary micromechanical air valve with and without pressure applied to the control channel. FIG. 11 is a schematic top view of a 5 × 1 array device with 5 pairs of air valves and a pair of electrodes. FIG. 12 is a schematic top view of a 5 × 1 array device using two upper electrodes. FIG. 13 is a schematic top view of a 2x1 array device with two pairs of air valves and two upper electrodes.

An exemplary embodiment will be described more fully with reference to the accompanying drawings.

FIG. 1 shows an apparatus 10 for producing one or more nanopores on the film 12. This device mainly consists of an upper (first) substrate 14, a lower (second) substrate 15, and a support 16 arranged between the upper substrate 14 and the lower substrate 15. The support 16 is configured to hold the dielectric thin film 12 that defines the opposing planes 13. For illustrative purposes, a single microfluidic channel 4 is formed on the upper substrate 14, and a larger commonly used microfluidic channel 5 is formed on the lower substrate 15. A pair of electrodes 17 electrically connected to the voltage source 18 is used, one electrode is placed on each of the microfluidic channels 4 and 5, and a potential difference is generated on both sides of the membrane. As further described below, the electrodes may be arranged differently to provide the device with more microfluidic channels than described above.

The device 10 further includes a current sensor (not shown) electrically connected to one of the electrodes, a current sensor and a controller 19 interfaced with a voltage source 18. During operation, the current sensor measures the current flowing through the membrane. Then, the controller 19 detects the sudden increase in the measured current, and in response to the detection of the sudden increase in the measured current, the potential difference generated on both sides of the film is generated as described further below. Make it disappear.

2A and 2B further show exemplary embodiments of device 10'. In this exemplary embodiment, an exposed SiN film (SiMPore) measuring 500 × 500 μm 2 and having a thickness of 20 nm.
Inc. A commercially available silicon chip (eg, frame size 3 mm) with SN100-A20Q05) served as the support 16 and was mounted between multiple microfluidic channel arrays of different architectures. Referring to FIG. 2A, the apparatus 10'presented herein utilizes a shape that includes five independently treatable microfluidic channels 21 on one side of the membrane 12 and on the opposite side of the membrane 12. Was communicated by one commonly used microchannel 22. Specifically, the apparatus 10'is an array of five independent microfluidic channels 21, which are the thickest channels (height 50 μm) with a width of 200 μm that gradually narrow to a width of 15 μm at the membrane 12, as best seen in FIG. 2B. Included. The five independent channels 21 are each 25 μm apart from each other. In this embodiment, five independent microfluidic channels have been shown, but it is easy to see that in other embodiments, more or fewer microfluidic channels may be formed.

In an exemplary embodiment, soft lithography uses polydimethylsiloxane PDMS (Sylgard 184, 7: 1 (w / w) ratio obtained from Dow Corning) patterned from a master mold created by soft photolithography. Each layer was prepared. In all configurations, the bottom layer consisted of a PDMS layer about 3 mm thick containing a single fluid channel 22 bonded to a glass slide (oxygen plasma adhesion, AutoGlow Research) 250 μm wide x 100 μm high. .. A 2 mm hole was manually drilled in the commonly used lower microchannel 22 on which the etched side of the silicon chip was placed so that the fluid could pass through to the nanopores. A thin layer of PDMS (100 ± 10 μm) was then spin-coated around the chip 16 to correct the thickness of the silicon chip, leaving a smooth, sealed surface on which multiple microfluidic channels could be bonded. After spin coating, the PDMS thin layer was cured on a hot plate at 80 ° C. for 20 minutes.

3A-3C further illustrate this fabrication process. The presented device integrates a commercially available silicon nitride (SiN) membrane (SN100-A20Q05, SiMPore Inc.) in a microfluidic device made of polydimethylsiloxane (PDMS). The PDMS layer was prepared by soft lithography and replicated from a master mold made of SU8-2050 photoresist (Microchem Inc.) on a silicon wafer. Each microfluidic layer (microfluidic vias, independent channel layer) with different spin rates, firing times and temperatures, UV exposure and developing times, depending on the final desired thickness (height) of the features obtained. And a commonly used channel layer).

Following the fabrication of each master mold, the wafer was first treated with aminosilane to facilitate the removal of PDMS. Next, PDMS (7: 1 (w / w) base for all layers: hardener) was poured into the master mold for each channel layer, followed by degassing in a vacuum chamber for 30 minutes and at 80 ° C. for 2 hours. It was fired. Then, the cured PDMS was released from a mold to prepare a microchannel structure. The individual device components were then cut out and perforated with access holes for fluid and electrode introduction in independent channels (outer diameter 0.75 mm for fluid tubes, outer diameter 1.25 mm for electrodes). A 2.0 mm hole was manually drilled in the center of the commonly used microchannel to allow fluid to pass through the bottom of the chip. With reference to FIG. 3A, oxygen plasma (Glow Research AutoGlow) was used to bond the silicon chips (etched side) to the common channel layer above the perforated holes. All plasma bonding steps were performed at 30 W for 30 seconds.

PDMS to correct the thickness of the silicon chip and leave a flat, smooth surface for bonding independent (upper) channels in both configurations (with and without the microfluidic via layer). A thin layer (approximately 100 ± 10 μm) was spin coated around the chip (500 rpm for 5 seconds, followed by 1000 rpm for 10 seconds). The thin layer was placed directly on a hot plate and cured at 80 ° C. for 20 minutes.

A fluidly separated upper side prior to bonding the Ag / AgCl electrode to the PEEK tube through which the electrolyte (or ionic) solution flows so that fluid can pass through the microfluidic channel and conduct electricity. Holes were drilled in each of the channels in and the lower commonly used channels. By placing the electrode approximately 5 mm from the center of the membrane, the resistance of the microchannel to the nanopores is approximately 100 kΩ in a 1 M KCl electrolyte solution, i.e. less than approximately 1% of the total electrical resistance of the device containing the nanopores 10 nm in diameter. Limited to. Finally, the commonly used channels were glued to a clean glass slide. Although the specific fabrication technique has been mentioned above, it can be understood that other lithography techniques are also included in the scope of the present disclosure.

Immediately before introducing the electrolyte solution into the microfluidic channel, the assembled device was treated with oxygen plasma at 70 W for 5 minutes to increase the hydrophilicity of the microchannel. The microfluidic channel was then connected to the sample vial with a polyethylene tube and the vial was pressurized using a precision pressure regulator to initiate the flow. By running a 1M KCl solution (pH 7.5) and attempting to measure the ion currents between the microfluidic channels under a moderate applied voltage (eg 0.2V to 1V), the microfluidic channels are prior to fabrication of the nanopores. An effective seal between them (> 10 GΩ) was tested.

Contaminations and monomers need to be removed from the microfluidic materials used in the manufacture of the device in order to improve the functionality of the device. In particular, polydimethylsiloxane (PDMS) pieces should be chemically treated with a solvent prior to device assembly, and plasma treatment can be used to remove contaminants on the surface of the membrane as a result of microfluidic integration. it can.

According to one aspect of the present disclosure, placing electrodes in the microfluidic channel should lead to a uniform electric field over the entire area of the insulating film. Various electrode arrangements can be used, depending on the microfluidic architecture as seen in FIGS. 4A-4D. When a single microchannel is placed in a thin insulating film, a pair of electrodes placed on one side of the film somewhere downstream of the microfluidic channel will generate a non-uniform electric field over the entire membrane surface. However, if two electrodes biased to the same potential in the same microfluidic channel are placed on either side of the membrane (ie, one electrode is upstream of the membrane and the other electrode is downstream of the membrane), the most in FIG. 4A. As is often the case, the uniformity of the electric field can be increased. In this example, a set of electrodes 30 is used to generate a potential difference on both sides of the film 12. A set of electrodes 30 includes a reference electrode 33 located below the membrane and two or more additional electrodes 32 located above the membrane. Specifically, the two electrodes 32 are arranged in the microfluidic channel of the upper substrate, while the reference electrode 33 is arranged in the commonly used microfluidic channel of the lower substrate. The two additional electrodes 32 are arranged with respect to the membrane so that the electric field throughout the membrane is uniform. For example, one of the additional electrodes 32 may be located upstream of the membrane and the other of the additional electrodes 32 may be located downstream of the membrane. Other arrangements of the two additional electrodes are also contemplated by the present disclosure.

Referring to FIG. 2A, the underside of the support 16 includes a tapered recess 13 that helps shape the electric field uniformly, similar to the role played by the vias, which allows a single reference electrode 33 to be used.

In some embodiments, the membrane 12 may be placed directly on the lower substrate 15 and supported by this substrate without the use of a support 16. In these embodiments, the second reference electrode 33 can be placed underneath the membrane, as seen in FIG. 4C. In particular, one reference electrode 33 may be located upstream of the membrane and the other two reference electrodes 33 may be located downstream of the membrane. In this way, the two reference electrodes 33 function to uniformly form the electrode field close to the film.

FIG. 4B shows another electrode arrangement. In this arrangement, a single electrode 35 is arranged in the looped microfluidic channel 36 containing the ionic solution in order to achieve a similarly uniform electric field over the entire membrane surface. The microfluidic channel 36 forms a loop downstream of the electrode 35, and a part of the loop passes over the membrane. The valves of such devices are pressurized and close the lower flow path. During the nanopore fabrication process, valve pressure will be released (microfluidic channels will be opened). Thus, due to the presence of the electrolyte solution through the looped channels, the electric field is uniformly formed. The above configuration can be scaled for several microfluidic channels using microvalve technology (eg, as seen in FIG. 11). In this alternative configuration, a single reference electrode 33 may be placed under the membrane as described in the context of FIG. 4A, or two reference electrodes may be placed under the membrane as described in the context of FIG. 4B. It is understood that it may be used.

Similarly, microelectrodes can be patterned with microfluidic channels to achieve a uniform electric field. These surface-patterned electrodes are held at the same potential and can be arranged as described above to produce a uniform electric field. A circular electrode centered on the insulating film can also guarantee the uniformity of the electric field. A single patterned microelectrode can be patterned directly above the insulating film or within each of the individual microfluidic channels. Such surface-patterned electrodes will be particularly useful for custom-designed chips where large arrays of nanopores may be formed. Other modifications of the electrode arrangement that produce a uniform electric field are also contemplated by the present disclosure.

In another aspect of the present disclosure, microvias can be added to the microfluidic system to facilitate the formation of electric fields in and around the vias. 5A and 5B show a second exemplary embodiment of the device 10 ″. In this embodiment, the apparatus is also composed of an upper substrate 14, a lower substrate 15, and a support 16 arranged between the upper substrate and the lower substrate. The support 16 is similarly configured to hold the dielectric thin film 12 defining the opposing planes 13. In this embodiment, an intermediate layer 19 is formed on the support 16 and is arranged between the upper substrate 14 and the support 16. One or more vias 51 may be formed in the intermediate layer 19 so as to generate a uniform electric field in and around the vias.

This second microfluidic configuration further reduces high frequency electrical noise by localizing CBD nanopore formation at each microchannel in the center of the membrane and minimizing the area of the membrane exposed to the ionic solution. Designed to In this second configuration, a 200 μm thick PDMS layer with an array of rectangular openings with a constant width of 15 μm and varying lengths from 40 μm to 120 μm is used to adequately cover the center of the membrane. A microfluidic via connecting the microfluidic channels was formed in the defined area. To create a thin (200 μm) microfluidic via layer to which independent channels can be adhered, degassed PDMS is spin-coated on its master mold (500 rpm for 5 seconds, followed by 800 rpm for 10 seconds) and directly onto the hot plate. It was placed and cured at 80 ° C. for 30 minutes. All alignment steps were performed using an OAI DUV / NUV mask aligner (model 206) to accurately place the channel layer independent of the microfluidic vias on the SiN membrane. This layer was then glued to an array of five independent PDMS microfluidic channels as in the initial design. Except for the above points, a second embodiment of apparatus 10 ″ was made in the same manner as described in the context of FIGS. 3A-3C.

To understand the effect of adding a microvia layer to the microfluidic configuration, we investigated a finite element model of the electric field in both device geometries (with and without microfluidic vias). Create the device configuration in 2D and COMSOL Multiphysics
The electric field was modeled using a computer study in the Electrical Currants module of Modeling Software. Both shapes were examined first with a complete membrane (ie, no aqueous solution flow through the membrane) and then with nanopores (with a 20 nm fluid conduit in the membrane).

FIG. 6A shows the shape of the device in which independent microchannels are placed directly on the membrane, and FIG. 6B shows the device containing microfluidic vias. Both devices contain a 20 nm pore in the center of the membrane. From the enlarged view of the region surrounding the nanopores in FIG. 6D, it can be seen that the electric field in the immediate vicinity of the nanopores in the microfluidic via configuration is relatively uniform between the membrane and the pores. This is accentuated by the fact that the strength of the electric field decays uniformly from the nanopores on either side of the membrane. Furthermore, the electric field lines are symmetrical from left to right, even though both electrodes are located 3 mm to the left of the nanopore. On the other hand, FIG. 6C shows that under the same conditions, the electric field lines are completely non-uniform in devices without microfluidic vias. Both field lines and field intensities differ from left to right in the entire membrane as well as in independent (upper) microchannels.

Further study of the shape of the electric field in these configurations reveals that nanopore fabrication using CBD can also be affected by asymmetric electrode placement. FIG. 6E shows the magnitude of the electric field through the horizontal cross section of the complete membrane in devices with microfluidic vias and devices without microfluidic vias. In this example, a voltage of 10 V across the membrane was applied to simulate the nanopore fabrication conditions actually used. Devices containing microfluidic vias exhibit a uniform electric field over the length of the exposed membrane, whereas devices with independent (upper) microchannels placed directly on the membrane are closer to the side where the electrodes are placed. Shows a moderately strong electric field.

In both exemplary embodiments, individual nanopores were made by inducing dielectric breakdown events at each of the independent microfluidic channels integrated over the membrane. Briefly, this was done by applying a high electric field using a custom-built electronic circuit. A voltage of 10V-14V with a commonly used grounded microchannel was applied to one of the independent microfluidic channels to create nanopores in minutes or seconds. The potential difference on both sides of the membrane produces an electric field with a value greater than 0.1 volt per nanometer. This voltage also induced a leak current through the SiN film. This is monitored in real time (see FIG. 7A). The formation of a single nanopore is detected by a sudden sudden increase in leakage current above a predetermined threshold, which cuts off the applied voltage with a response time of 0.1 seconds. Although threshold currents and response times can be varied to achieve the desired nanopore size following a disruption event, the sizes described herein are typically less than 2 nm in diameter (tight cuts). Condition). This process is then repeated in each of the upper fluidly separated microchannels to form nanopores that can be treated independently in different microfluidic channels on a single membrane. After fabrication of the nanopores, sensitive measurements of electrical characterization and single molecule sensing were performed using an Axopatch 200B (Molecular Devices) low noise current amplifier.

Heights formed by making each nanopore as described above and alternating -5V and + 5V pulses on both sides of the membrane to obtain nanopores of the desired size for detecting a particular biomolecule. Adjusted using an electric field. This process is used to optimize electrical noise characteristics and then recover the clogged nanopores for further experiments with results comparable to those reported in previous studies with macroscopic fluid reservoirs. Further details regarding this conditioning technique can be found in US Patent Application Publication No. 2015/0109088, wherein the title of the invention is "Method for Controlling the Size of Solid-State Nanopores", the entire contents of which are incorporated herein by reference. To do.

To estimate the diameter of each nanopore produced by CBD, the conductance G of the nanopores is measured directly in solution by monitoring the ionic current passing through each nanopore as the applied voltage is swept from -200 mV to +200 mV. did. By assuming a cylindrical shape and considering the access resistance 30, the effective diameter d of the nanopore can be calculated from its conductance by the following relational expression.

In Equation 1, σ is the bulk conductivity of the electrolyte and L is the effective length of the nanopores, which is assumed to be equal to the nominal thickness of the SiN film. The current-voltage (IV) curve of FIG. 2 (c) shows 1 M KCl (pH 7.5) for five independently formed nanopores of size 3 nm to 10 nm in a single 5-channel device. The ohmic response at (σ = 10.1 ± 0.1Sm-1) is shown. The error caused by ignoring the contribution from the surface charge of Equation 1 affects the accuracy of the calculated effective diameter of the nanopores by <0.5 nm at the high salinity used here, and the electrolyte conductivity and film. The error due to the thickness value affects the uncertainty of the nanopore diameter by about 0.3 nm.

To further characterize the performance, power spectral density plots (PSDs) of ion currents were obtained for nanopores made with each of the two microfluidic architectures (see Figure 8A). Low frequency noise (less than 1 kHz) is generally 1 / f type, but high frequency noise depends on the dielectric properties and capacitance of the device that occurs in the surface area exposed to the electrolyte solution. Therefore, minimizing the surface exposed to the solution leads to a reduction in this high frequency noise and a significant improvement in the signal-to-noise ratio during biomolecule sensing at high bandwidths. This is shown in FIG. 8A. Here, both 5-channel devices (with and without microvias) are compared to nanopore chips mounted between fluid reservoirs in standard macrofluid cells. At this high frequency range, 5-channel microfluidic devices (without microvias) exhibit noise characteristics comparable to those obtained with macroscopic cells. This result argues that the noise in this regime is due to the amount of exposed area of the membrane, calculated to be about 3 x 105 μm2 for the macro reservoir and about 2 x 105 μm 2 for the microchannel of a standard 5-channel device. Match. However, using the smallest microvia (40 x 15 μm 2) of a 5-channel device, the high frequency noise is significantly reduced when the exposed area of the film is reduced to about 1/350, about 6 x 102 μm 2. This noise reduction is further accentuated by the baseline ion current traces of each device without the voltage shown in FIG. 8B, where peak-to-peak noise in the 100 kHz bandwidth is halved in configurations with microvia. It is reduced to 1 (5 at 10 kHz bandwidth), and RMS noise is reduced to 1/7 at 10 kHz and 1/2 at 100 kHz bandwidth.

The functionality of these devices was evaluated by observing the movement of biomolecules with reference to FIGS. 9A and 9B. In each case, as described above, nanopores were first prepared and expanded to the desired diameter. After introduction of the sample, the flow in the microfluidic channel was minimized by turning off the pressure regulator. FIG. 9A shows individual human α-thrombin (Haematological Technologies, Inc.) molecules at 250 μM concentrations detected at 1 M KCl (pH 8.0) using 10.5 nm nanopores of the microfluidic channel (without vias). A scatter plot of the cutoff of conductance and the period at that time is shown. Here, protein molecules were loaded into one of the five independent upper microfluidic channels and biased at −200 mV against the grounded lower commonly used channel. Overall, over 5,000 individual events were observed. FIG. 9B shows a similar scatter plot for DNA transfer events through different nanopores of 11.5 nm localized in microchannels containing microvias. Here, a 3 pM solution of 10 kbp dsDNA in 2 M KCl (pH 10) is added to the upper microchannel and biases of -200 mV, -250 mV, -300 mV are applied to the commonly used channels to 1,500. Got more than a move event. It is noteworthy that the conductance block sizes obtained at both the protein and single-level dsDNA events are consistent with previously reported models and experiments using standard macrofluid cells.

Microfluidic design must be carefully considered when integrating nanopores using this technique. Integrated nanopores (without microvias) in microfluidic channels placed directly on the membrane capture and detect proteinaceous samples in 30% (9 out of 30) of the devices tested. However, the capture efficiency and experimental yield of devices capable of exhibiting nucleic acid migration were significantly reduced. Here, the criterion used to define the experimental yield is a device capable of detecting more than 1000 biomolecule migration events. It is important to note that placing electrodes in a microfluidic channel leading to the membrane causes electric field heterogeneity near the nanopores and in the membrane if the upper microchannel contains only a single electrode. Due to this asymmetry, nanopores may be formed near the edge of the membrane (near the edge of the silicon support chip), which is a region that is more susceptible to stress during adhesion to the PDMS microchannel layer. In this region, due to the surface charge properties of the membrane near the nanopores, the movement of highly charged large nucleic acid polymers may be electrostatically impeded while allowing less charged polypeptides to pass through. However, with the introduction of microvia, nanopore fabrication is localized to the intended region away from the center or edges of the membrane, resulting in a more symmetrical electric field in three of the four devices tested at pH 10. As mentioned above, it is also possible to reduce this asymmetry of the electric field by incorporating a pair of electrodes biased at the same potential in the upper independent channel on one side of the membrane. In this configuration, 5 out of 6 devices tested at pH 8 succeeded in detecting at least 1000 biomolecule migration events.

In yet another aspect of the disclosure, microvalve technology can play a role in achieving large-scale integration of microfluidics. The development of functionally reliable microvalves is also an important step towards the successful miniaturization and commercialization of fully automated microfluidic systems. Microvalves are used to control the flow of fluid and to carry electrical / ion currents throughout the microfluidic network. Various techniques such as screw valves, pneumatic valves, solenoid valves, etc. can be used to incorporate valves in microfluidic devices.

FIG. 11 shows an exemplary embodiment of device 110 using pneumatically driven microvalve technology. The apparatus mainly comprises an upper substrate, a lower substrate, a support arranged between the upper substrate and the lower substrate, and includes an intermediate via layer as described in the above-described embodiment. You can also. In this exemplary embodiment, five microfluidic channels 112 are formed on the upper substrate. Again, in other embodiments, more microfluidic channels can be formed or a smaller number of microfluidic channels can be formed.

The microfluidic channel 112 is passed adjacent to the membrane so as to generate a uniform electric field over the entire region of the membrane. For example, each microfluidic channel 112 forms a loop downstream of the electrode 116, where a portion of the loop passes over the membrane. Different closed-loop configurations that provide electric field lines from two opposing sides of the membrane are also within the scope of the present disclosure.

The control valve 114 is also located on the microfluidic channel 112 and operates to control the conductive path determined by the open or closed valve in the channel. In an exemplary embodiment, the microfluidic channel 112 is embedded in an elastomeric polymer to achieve a pneumatically driven microvalve. These valves are generally made in two layers by using soft lithography techniques. With reference to FIGS. 10A and 10B, the valve is composed of two layers separated by a layer of very thin membrane, as shown in 108 of FIG. 10A. One layer (flow layer) 106 has a channel for passing a fluid. As shown in FIG. 10B, the separation thin film is distorted toward the microfluidic channel side when the control channel (valve) of the other layer (control layer) 107 is pressurized by air or water. As a result, the flow of the fluid (liquid electrolyte) is stopped, resulting in a sealed state. The amount of flow channel closure is related to the electrical impedance applied by the valve to the electrical network. For example, when the flow channel is completely closed, the impedance can be> 10 GΩ (the exact value depends on the conductivity of the electrolyte and the shape of the valve), effectively isolating that region of the microfluidic network.

Returning to FIG. 11, at least two valves 114 are arranged in each of the five microfluidic channels 112, and one valve is arranged on each side of the membrane. Further, each valve 114 is fluidly connected to an air pressure source (not shown) and is actuated by this air pressure source. By controlling the degree to which the valves in each group close the microfluidic channel 112, the valves 114 can act as variable resistors for the voltage divider. In this way, valves can be used to allow potentials to pass through selected microfluidic channels to generate a uniform electric field along the region of the membrane.

Including pneumatically driven microvalves is a practical way to achieve large-scale integration in microfluidics. It is a solid method of controlling the value of the potential difference on both sides of the membrane in each microchannel group independently on-chip with several electrodes. The microvalve acts as a voltage divider (providing a seal with resistance> 10 GΩ on the microchannel) that allows precise control of the electric field in various regions of the membrane. This control gives the ability to handle any number of nanopores for fabrication, size control, sensing, and redirects potential using a single electrode pair located somewhere in the fluid channel on either side of the membrane. Can be used to generate a uniform electric field along the length of the membrane on a particular microchannel (an important feature for biomolecule detection) using a single pair of electrodes and is commonly used. Required for array fabrication and sensing on devices containing microchannels (features required for series and parallel probing of single samples with multiple nanopores), for solutions containing various solvents, ionic strengths, pH or specimens. It is required for device scalability and functionality in that it allows for rapid exchange, facilitates fabrication and sensing, and includes variable fluid and electrical resistors for on-chip fabrication and biomolecule sensing. Preserving the hydrophobicity of the valve and channel cross sections is also important for obtaining high resistance seals used to control the magnitude and uniformity of the electric field throughout the membrane during fabrication and sensing. Please note. This is achieved by chemically treating each layer of the device prior to assembly, eliminating the need to plasma the membrane to remove contaminants that leave the valve cross section hydrophilic.

The control of these resistance valves can be utilized to bring different positions in the microfluidic network to specific potential conditions with a small number of electrodes. In this embodiment, a pair of electrodes can be used. Electrodes 116 are located in the microfluidic channels on both sides of the membrane (only the upper electrode is shown in FIG. 11, but the lower electrode is similarly located below the membrane). Except for the differences described above, device 110 is similar to the device described in the context of FIG. 2A.

12 and 13 show other exemplary embodiments of the device using pneumatically driven microvalve technology. In FIG. 12, device 120 is similar to device 110, but further includes a routing valve 121 and a second upper electrode 116. During operation, the routing valve 121 remains closed and the ionic solution flows through the channel from both the left and right sides of the membrane towards the membrane 12 as shown. The routing valve 121 actually creates two microfluidic subsystems. One electrode is located upstream where the channel is divided into five separate microfluidic channels in each of the two microfluidic subsystems.

FIG. 13 shows a similar device 130, but with only two microfluidic channels 112. Similarly, two upper electrodes are placed on either side of the membrane and two microfluidic channels pass through a portion of the membrane. Two control valves 114 are located in each microfluidic channel 112, one upstream of the membrane and one downstream of the membrane. Except for the differences described above, these two devices 120, 130 are similar to the devices described in the context of FIG.

The above description of the embodiments are provided for illustration and description. It is not intended to be exhaustive or to limit disclosure. The individual elements or features of a particular embodiment are usually not limited to that particular embodiment, but where applicable, they can be interchanged without specific illustration or description and selected embodiments. Can be used in. The same can be changed in many ways. Such modifications should not be considered as deviations from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

The terms used herein are for the purpose of describing particular exemplary embodiments only and are not intended to be limiting. As used herein, the singular "a," "an," and "the" are intended to include more than one, unless the context explicitly indicates otherwise. The expressions "comprise," "comprising," "inclusion," and "having" are comprehensive and identify including notational features, integers, steps, operations, elements and / or the presence of components, but one. It does not preclude the addition or existence of the above other features, integers, steps, behaviors, elements, components and / or groups thereof. The method steps, processes, and operations described herein should not necessarily be construed as requiring performance in the particular order described or illustrated, unless specifically specified as the order of performance. Please understand that additional or alternative steps may be used.

When one element or layer is said to be "on", "engaged", "connected" or "connected" to another element or layer, then another element or layer or intervening element or layer is present. May be good. In contrast, if one element is said to be "directly on top", "directly engaged", "directly connected" or "directly connected" to another element or layer, it intervenes. There is no element or layer to do. Other words used to describe relationships between elements should be interpreted in a similar manner (eg, "between" and "directly", "adjacent" and "directly adjacent"). As used herein, the term "and / or" includes any and all combinations of one or more of the related listed items.

Terms such as first, second, and third are used herein to describe various elements, components, areas, layers and / or sections, but these elements, components, areas, Layers and / or sections should not be limited to these terms. These terms may be used solely to distinguish one element, component, area, layer or section from another area, layer or section. "First", "second" and other numerical expressions do not imply order or order unless explicitly indicated by the context. Therefore, the first element, component, region, layer or section described below is referred to as the second element, component, region, layer or section without departing from the teaching content of the exemplary embodiment. be able to.

Space-related terms such as "inner,""outer,""beneath,""below,""lower,""above," and "upper" are associated with an element or function, as shown in the figure. It is intended to make it easier to explain the relationship to other elements or functions. Spatial relative terms may be intended to include different orientations of the device during use or operation, in addition to the orientations shown. For example, when the device in the figure is turned over, the elements described in "below (bottom)" or "beneath (bottom)" of other elements or functions are placed in "above (top)" of other elements or functions. Will be done. Thus, the term "below" can include both up and down directions. The device can be oriented in any other direction (rotate 90 degrees or in any other direction), and therefore the spatially relative descriptors used herein are interpreted accordingly. Will be done.

Claims (33)

A device for forming one or more nanopores on a film composed of at least one dielectric layer that defines opposing planes.
A first substrate with commonly used microchannels formed on the exposed surface,
A support arranged on the exposed surface of the first substrate and configured to hold the film, and
A second substrate in which one or more microfluidic channels are formed on the inner surface, wherein the one or more microfluidic channels are fluidly separated from the commonly used microchannels by the membrane. A second substrate arranged on the support so that the inner surface faces the support as described above.
A set of electrodes comprising a reference electrode disposed on one side of the membrane and two or more additional electrodes disposed on the opposite side of the membrane.
The set of electrodes creates a potential difference on both sides of the membrane, and the two or more additional electrodes are arranged relative to the membrane so that the electric field of the entire membrane is uniform. .. The device of claim 1, wherein the potential difference of the film produces the electric field having a value greater than 0.1 volt / nanometer. A current that is electrically connected to one of the electrodes and can operate to measure the current flowing between one of the one or more microfluidic channels and the commonly used microchannel. With the sensor
Further comprising a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of pores penetrating the membrane and the sudden increase in the measured current. The device according to claim 1, wherein the potential difference generated on both sides of the membrane disappears in response to the detection of the increase. The two or more additional electrodes were arranged in a first electrode located upstream of the membrane in the one or more microfluidic channels and downstream of the membrane in the one or more microfluidic channels. The device of claim 1, comprising a second electrode. 4. The set of electrodes comprises two reference electrodes arranged in the commonly used microchannel such that one is upstream of the membrane and the other is downstream of the membrane. Equipment. The first aspect of the present invention, wherein the support is formed in a region adjacent to the membrane and further includes a tapered recess fluidly connected to the commonly used microchannel provided on the first substrate. Equipment. A set of electrodes and a plurality of microfluidic channels formed on the inner surface of the second substrate are further provided, and a set of microchannels provided in the plurality of microfluidic channels are related to each other. The device according to claim 1, which has an electrode. An electric field that is an intermediate layer that is arranged directly on the support and is arranged between the support and the second substrate, in which at least one via is formed and is uniform in the vicinity of the via. The apparatus according to claim 1, further comprising the intermediate layer configured to generate the above. The device of claim 1, wherein the one or more microfluidic channels are on the order of microns. The device of claim 1, wherein the one or more microfluidic channels are on the order of nanometers. A device for forming one or more nanopores on a film composed of at least one dielectric layer that defines opposing planes.
A first substrate with commonly used microchannels formed on the exposed surface,
A support arranged on the exposed surface of the first substrate and configured to hold the film, and
An intermediate layer arranged on the support and formed with at least one via.
A second substrate in which one or more microfluidic channels are formed on the inner surface, wherein the one or more microfluidic channels are fluidly separated from the commonly used microchannels by the membrane. A second substrate arranged on the intermediate layer so that the inner surface faces the support as described above.
A pair of electrodes arranged on the front and back surfaces of the film,
The pair of electrodes generate potential differences on both sides of the membrane, and the at least one via in the intermediate layer fluidly connects the one or more microfluidic channels to the exposed surface of the membrane and said vias. A device that is configured to generate a uniform electric field in and around it. The device of claim 11, wherein the potential difference of the film produces the electric field having a value greater than 0.1 volt / nanometer. A current that is electrically connected to one of the electrodes and can operate to measure the current flowing between one of the one or more microfluidic channels and the commonly used microchannel. With the sensor
Further comprising a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of pores penetrating the membrane and the sudden increase in the measured current. The device according to claim 11, wherein the potential difference generated on both sides of the membrane disappears in response to the detection of the increase. The device of claim 10, wherein the depth of the at least one via is greater than the diameter of the at least one via. The second substrate comprises an array of microfluidic channels formed on the inner surface, each of which is aligned with one of the microfluidic channels in the array of microfluidic channels. The device according to claim 10, wherein the apparatus includes a plurality of vias in this manner. A device for forming one or more nanopores on a film composed of at least one dielectric layer that defines opposing planes.
A first substrate with commonly used microchannels formed on the exposed surface,
A support arranged on the exposed surface of the first substrate and configured to hold the film, and
A second substrate in which one or more microfluidic channels are formed on the inner surface, wherein the one or more microfluidic channels are fluidly separated from the commonly used microchannels by the membrane. A second substrate arranged on the support so that the inner surface faces the support as described above.
A pair of electrodes arranged on the front and back surfaces of the film,
The pair of electrodes generate potential differences on both sides of the membrane, and the one or more microfluidic channels pass adjacent to the membrane so as to generate a uniform electric field over the region of the membrane. There is a device. One of the pair of electrodes is arranged in the one or more microfluidic channels, the one or more microfluidic channels form a loop downstream of the electrode, and a part of the loop is on the membrane. The device of claim 16, which passes through. 16. The apparatus of claim 16, further comprising a control valve arranged in the one or more microfluidic channels and operating to control the amount of flow through the one or more microfluidic channels. The device of claim 18, wherein the control valve is fluidly coupled to the pneumatic source and further defined as an elastomeric polymer actuated by the pneumatic source. The second substrate comprises an array of microfluidic channels formed on its inner surface, with each microfluidic channel of the array of microfluidic channels passing through a portion of the membrane and at least two control valves. 16. The device of claim 16, wherein the device is located internally, one valve located upstream of the membrane and the other valve located downstream of the membrane. 20. The apparatus of claim 20, wherein the value of the potential difference of the membrane is controlled by adjusting the flow through the control valve arranged in the array of microfluidic channels. A device for producing one or more nanopores on a membrane.
Commonly used microchannels are arranged on the exposed surface of the first substrate and the first substrate formed on the exposed surface, demarcate facing planes, and consist of at least one dielectric layer. Membrane and
A second substrate in which one or more microfluidic channels are formed on the inner surface, wherein the one or more microfluidic channels are fluidly separated from the commonly used microchannels by the membrane. With the second substrate arranged on the film so that the inner surface faces the film as described above.
A set of electrodes comprising two reference electrodes disposed on one side of the membrane and two or more additional electrodes disposed on opposite sides of the membrane.
A device in which the set of electrodes is arranged with respect to the film so that a potential difference is generated on both sides of the film and the electric field of the film is uniform. 22. The apparatus of claim 22, wherein the potential difference of the film produces the electric field having a value greater than 0.1 volt / nanometer. A current that is electrically connected to one of the electrodes and can operate to measure the current flowing between one of the one or more microfluidic channels and the commonly used microchannel. With the sensor
Further comprising a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of pores penetrating the membrane and the sudden increase in the measured current. 22. The apparatus of claim 22, which eliminates the potential difference generated on either side of the membrane in response to detection of an increase. The two additional electrodes are a first electrode arranged in the one or more microfluidic channels upstream of the membrane and a second electrode arranged in the one or more microfluidic channels downstream of the membrane. 22. The apparatus of claim 22, comprising. 25. The apparatus of claim 25, wherein the two reference electrodes are arranged in the commonly used microchannel such that one is upstream of the membrane and the other is downstream of the membrane. A plurality of microfluidic channels formed on the inner surface of the second substrate and a plurality of sets of electrodes are further provided, and a set of microchannels provided in the plurality of microfluidic channels are associated with each other. 22. The device of claim 22, which has an electrode. A device for producing one or more nanopores on a membrane.
Commonly used microchannels are arranged on the exposed surface of the first substrate and the first substrate formed on the exposed surface, demarcate facing planes, and consist of at least one dielectric layer. Membrane and
An intermediate layer arranged on the membrane and formed with at least one via,
A second substrate in which one or more microfluidic channels are formed on the inner surface, wherein the one or more microfluidic channels are fluidly separated from the commonly used microchannels by the membrane. A second substrate arranged on the intermediate layer so that the inner surface faces the support as described above.
A pair of electrodes arranged on the front and back surfaces of the film,
The pair of electrodes generate potential differences on both sides of the membrane, and the at least one via in the intermediate layer fluidly connects the one or more microfluidic channels to the exposed surface of the membrane and said vias. A device that is configured to generate a uniform electric field in and around it. 28. The apparatus of claim 28, wherein the potential difference of the film produces the electric field having a value greater than 0.1 volt / nanometer. A current that is electrically connected to one of the electrodes and can operate to measure the current flowing between one of the one or more microfluidic channels and the commonly used microchannel. With the sensor
Further comprising a controller interfaced with the current sensor, the controller detects a sudden increase in the measured current indicating the formation of pores penetrating the membrane and the sudden increase in the measured current. 28. The apparatus of claim 28, which eliminates the potential difference generated on either side of the membrane in response to detection of an increase. 28. The apparatus of claim 28, wherein the depth of the at least one via is greater than the diameter of the at least one via. The second substrate comprises an array of microfluidic channels formed on the inner surface, each of which is aligned with one of the microfluidic channels in the array of microfluidic channels. 28. The device of claim 28, thus comprising a plurality of vias. A device for forming one or more nanopores on a film composed of at least one dielectric layer that defines opposing planes.
A first substrate with commonly used microchannels formed on the exposed surface,
A support arranged on the exposed surface of the first substrate and configured to hold the film, and
A second substrate with an array of microfluidic channels formed on the inner surface, said inner so that the array of microfluidic channels is fluidly separated from the commonly used microchannel by the membrane. Each microfluidic channel in the array of microfluidic channels passes through a portion of the membrane, one upstream of the membrane and the other A second substrate with at least two control valves internally located downstream of the membrane.
A pair of electrodes arranged on the front and back surfaces of the film,
The pair of electrodes is a device that generates a potential difference on both sides of the film.

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